Thyroid hormone receptor-β1 signaling is critically involved in regulating secondary ossification via promoting transcription of the Ihh gene in the epiphysis

Abstract

Thyroid hormone (TH) action is mediated through two nuclear TH receptors, THRα and THRβ. Although the role of THRα is well established in bone, less is known about the relevance of THRβ-mediated signaling in bone development. On ther basis of our recent finding that TH signaling is essential for initiation and formation of secondary ossification center, we evaluated the role of THRs in mediating TH effects on epiphysial bone formation. Two-day treatment of TH-deficient Tshr^−/− mice with TH increased THRβ1 mRNA level 3.4-fold at day 7 but had no effect on THRα1 mRNA level at the proximal tibia epiphysis. Treatment of serum-free cultures of tibias from 3-day-old mice with T₃ increased THRβ1 expression 2.1- and 13-fold, respectively, at 24 and 72 h. Ten-day treatment of Tshr^−/− newborns (days 5–14) with THRβ1 agonist GC1 at 0.2 or 2.0 μg/day increased BV/TV at day 21 by 225 and 263%, respectively, compared with vehicle treatment. Two-day treatment with GC1 (0.2 μg/day) increased expression levels of Indian hedgehog (Ihh) 100-fold, osterix 15-fold, and osteocalcin 59-fold compared with vehicle at day 7 in the proximal tibia epiphysis. Gel mobility shift assay demonstrated that a putative TH response element in the distal promoter of mouse Ihh gene interacted with THRβ1. GC1 treatment (1 nM) increased Ihh distal promoter activity 20-fold after 48 h in chondroctyes. Our data suggest a novel role for THRβ1 in secondary ossification at the epiphysis that involves transcriptional upregulation of Ihh gene.

endochondral ossification is one of two essential processes required for fetal development of the mammalian skeletal system and for fracture healing. Unlike intramembranous ossification, which is the other process by which mesenchymal cells from the cranial neural crest, sclerotomes, and lateral plate mesoderm migrate and proliferate, forming mesenchymal condensations, cartilage is present and replaced by trabecular bone during endochondral ossification (16, 35). There are two centers of ossification for endochondral ossification, a primary and a secondary center. The primary ossification center (POC) usually appears in the diaphysis of the long bones or in the body of the irregular bones during prenatal development while the secondary ossification center (SOC) occurs in the epiphysis of long bones at the time of birth in mammals (11). Endochondral ossification at the POC is tightly regulated by a number of growth factors (PTHrp, IHH, IGF-I, BMP/TGFβ, Wnt, VEGF) and transcription factors (Sox9, Runx2, osterix, β-catenin) (3, 10, 21–23, 29, 42, 53). Dysregulation in the production and/or actions of any of the factors that regulate endochondral ossification can result in skeletal diseases including chondrodysplasias and osteoarthritis (21, 40). Although the processes leading to POC formation have been well established, signaling pathways that stimulate SOC formation are not well understood.

Thyroid hormone (TH) is known to play an important role in normal endochondral ossification and is essential for skeletal development, linear growth, maintenance of bone density, and efficient fracture healing (20, 45, 47, 48). Juvenile hypothyroidism causes dwarfism with delayed bone formation and mineralization, while TH replacement induces rapid catch-up growth (1, 20, 44, 45). Mice with targeted disruption of TH receptors (THRs) exhibit reduced bone length and bone mass (5, 12, 13, 44). By contrast, childhood thyrotoxicosis accelerates bone formation with premature closure of the growth plates and skull sutures, leading to short stature and craniosynostosis (15, 19, 37). The increase in TH levels during the prepubertal growth period is obligatory for the increase in IGF-I expression and trabecular bone mass during this period in mice (47, 48). In our recent studies on mechanisms for the TH effect, we focused on SOCs, since the time of appearance of SOCs in several species including mice, rats, and humans coincides with the time when peak levels of TH are attained (7, 17, 24). In humans, the identification of proximal humeral epiphsyial ossification centers occurs around a gestational age of 38 wk, which coincides with the attainment of peak levels of TH (36–40 wk). In rodents, the peak levels of TH are attained at week 2 when SOC formation also takes place. By using mouse models that are deficient in TH and growth hormone, we found that endochondral ossification of SOC is severely compromised due to TH deficiency and that TH treatment for 10 days completely rescued this phenotype (47). The conversion of cartilage into bone failed to occur in the TH-deficient mice, while much of the cartilage in the epiphysis was converted into bone in the control newborns that had normal TH levels. Immunohistochemistry studies revealed that TH treatment of thyroid-stimulating hormone receptor-deficient (Tshr^−/−) mice induced expression of Indian hedgehog (Ihh) and osterix (Osx) in collagen 2 (Col2)-expressing chondrocytes in the SOC at day 7, which subsequently differentiated into collagen 10 (Col10)/osteocalcin-expressing chondroosteoblasts at day 10. These studies indicate that TH regulates the SOC initiation and progression via differentiating chondrocytes into bone matrix, producing osteoblasts by stimulating Ihh and Osx expression in chondrocytes (47).

TH effects are known to be mediated via THRα and THRβ. Of the two receptors, THRα1 is severalfold more abundant than THRβ1 in both osteoblasts and chondrocytes (2, 25); THRβ2 is barely detectable in bone. Furthermore, studies have shown that bone growth is severely affected in mice with disruption of THRα1 (5). Although the role of THRα1 is well established, less is known about the relevance of THRβ-mediated signaling in bone development. In this study, we demonstrated expression and function of THRβ1 during the prepubertal growth period and found that THRβ1 in the presence of triiodothyronine (T₃) binds to a TH response element (TRE) in the distal Ihh promoter, stimulates Ihh transcription, and promotes endochondral bone formation in the epiphysis.

MATERIALS AND METHODS

Chemicals, cell lines, and biological reagents.

T₃ and T₄ were purchased from Sigma (St. Louis, MO). Anti-6xhis antibody was purchased from RandD Systems (cat. no. MAB050, Clone AD1.1.10, Minneapolis, MN). GC1 was a gift from Prof. Thomas S. Scanlan (Oregon Health and Sciences University, Portland, OR). [γ-³²P]ATP was purchased from PerkinElmer Life Sciences (Waltham, MA). pTAL-SEAP (secreted alkaline phosphatase) and Great EscAPe plasmids, and SEAP detection kits were from Clontech (Mountain View, CA). The pTAL-Ihh SEAP plasmid was generated by inserting a 1-kb portion of the mouse Ihh promoter (−4920 to −5919) in front of the TK minimal promoter of the pTAL-SEAP reporter. The chondrogenic cell line ATDC5 derived from teratocarcinoma AT805 was purchased from the American Type Culture Collection (Manassas, VA).

Mouse models.

Tshr^−/+ heterozygous mice with a point mutation in the coding region of the TSHR gene (Tshr) were purchased from the Jackson Laboratory (Bar Harbor, ME). DNA extracted from tail snips were used for PCR-based genotyping. Mice were housed at the VA Loma Linda Healthcare System Veterinary Medical Unit (Loma Linda, CA) under standard approved laboratory conditions. All the procedures were performed with the approval of the Institutional Animal Care and Use Committees of the VA Loma Linda Healthcare System. Mice were anesthetized with approved anesthetics (isoflurane, ketamine-xylazine) prior to procedures. For euthanasia, animals were exposed to CO₂ prior to cervical dislocation.

TH replacement.

Tshr^−/− newborn mice were intraperitoneally injected with a combination of 1 μg of T₃ and 10 μg of thyroxine (T₄) daily for 2 days (days 5 and 6) as described previously (47). Mice were injected daily for 2 days with 0.2 or 2.0 μg GC1 for 2 days (days 5 and 6) or 10 days (days 5–14). The doses selected were based on previously published in vivo studies on GC1 as well as our studies on the TH effects on bone (14, 47). The same volume of vehicle (5 mM NaOH) was given to control mice. At different times after initiation of TH treatment, mice were euthanized, and bones were removed and used for various analyses.

Micro-CT evaluation of the secondary ossification centers.

Trabecular bone microarchitecture of the tibia epiphyses (i.e., the SOC) isolated from 21-day-old mice were assessed by μCT (viva CT40; Scanco Medical, Switzerland) as described previously (49, 51). The tibias were fixed in 10% formalin overnight, washed with PBS, and immersed in PBS to prevent them from drying. The bone was scanned by X-ray at 55 kVp volts at a resolution of 10.5 μm/slice for trabecular microarchitecture assessment. The scout view of the whole leg, including the tibia epiphysis and the femur epiphysis was used for analysis. To analyze SOC formation, the proximal tibia epiphyses were used for measurement of newly formed bone. Parameters such as bone volume (BV, mm³), bone volume fraction (BV/TV, %), trabecular number (Tb.N, mm⁻¹), trabecular thickness (Tb.Th, μm) and trabecular space (Tb.Sp, μm) were evaluated as described previously (6, 49, 51).

Immunohistochemistry analyses.

Immunohistochemistry was performed using a rabbit immunohistochemistry kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instruction. Briefly, tibial epiphysial sections were deparaffinized in Histochoice clearing agent, rehydrated in a graded series of ethanol and tap water, and treated with 3% H₂O₂ for 30 min to inactivate endogenous peroxidase activity. The sections were then rinsed thoroughly with PBS (pH 7.4) and heated for 20 min at 90°C in sodium citrate-citric acid buffer (pH 2.5) for epitope recovery. The sections were pretreated with a blocking solution containing normal goat serum for 20 min and then incubated with primary antibodies against THRβ1 at dilution of 1:50 (Santa Cruz, sc-738). Negative control sections were incubated with normal rabbit IgG. After overnight incubation at 4°C, the sections were rinsed with PBS and incubated with biotinylated anti-rabbit secondary antibodies for 30 min at room temperature. The sections were then washed in PBS, incubated with VECTASTAIN Elite ABC Reagent for 30 min, rinsed again with PBS, and incubated with the Vector Blue substrate until the desired color stain developed.

Cell culture.

ATDC5 cells were maintained in DMEM-F12 medium containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml), as described (47). Cells were incubated in the presence of serum-free DMEM-F12 medium containing 0.1% bovine serum albumin (BSA) and antibiotics for 24 h prior to treatment with T₃ or vehicle. At different times, cultures were terminated and used for RNA extraction and real time PCR analyses.

Mouse epiphysis bone culture.

Tibial epiphyses were surgically isolated from 3-day-old C57BL/6 mice and were incubated in serum-free α-MEM containing 0.5% BSA, 50 μg/ml ascorbic acid, 1 mM β-glycerol phosphate, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in humidified air with 5% CO₂, as reported (27). T₃ (10 ng/ml) or the same volume of vehicle control was added to medium 1 day later, and bones were cultured for an additional 2 days or 10 days and subsequently used for RNA extraction or μCT analyses.

RNA extraction and quantitative PCR.

RNA was extracted from ATDC5 cells or primary chondrocytes as described previously (49). Epiphyses and growth plate regions of long bones were isolated and ground to powder in liquid nitrogen using a mortar and pestle prior to RNA extraction (48). An aliquot of RNA (25 ng) was reverse-transcribed with an oligo(dT)_12–18 primer into cDNA in a 20-μl reaction volume. The real-time PCR reaction contained 0.5 μl of template cDNA, 1× SYBR Green Master Mix (ABI), and 100 nM of specific forward and reverse primers in a 25-μl reaction volume. Primers for peptidyl prolyl isomerase A (PPIA) were used to normalize the expression data for the genes of interest. The primer sequences used for real-time PCR are listed in Table 1.

Table 1.

Antibody table

Peptide/Protein Target	Antigen Sequence (if known)	Name of Antibody	Manufacturer, Catalog No., and/or Name of Individual Providing the Antibody	Species Raised In; Monoclonal or Polyclonal	Dilution Used
THRβ1		Anti-THRβ1	Santa Cruz sc-738	Polyclonal antibody from rabbit	1:50 for IHC
6x Histidine	6x his	Anti-6x his monoclonal antibody	R&D Systems cat. no. MAB050, Clone AD1.1.10	monoclonal antibody from mouse	1 μg/20 μl volume reaction in gel supershift assay

THR, thyroid hormone receptor.

SEAP reporter assay.

The pTAL-Ihh SEAP and control pTAL-SEAP reporter constructs were transfected into ATDC5 cells by electroporation as reported previously (52). Briefly, ATDC5 cells were grown to 80% confluence before trypsin digestion. The cells (1.5 × 10⁶) were resuspended in 100 μl of fibroblast nucleofector buffer (Amaxa, Gaithersburg, MD) containing 8 μg of pTAL-Ihh SEAP or control plasmid and with 4 μg of the pcDNA3-THRβ1 construct. The cells were then transferred into a 2-mm gap width electroporation cuvette and electroporated at 165 V for 15 ms, using a Gene Pulser (Bio-Rad, Hercules, CA). After electroporation, the cells were equally plated in a 24-well plate in prewarmed α-MEM containing 5% double charcoal stripped FBS and cultured in a humidified 37°C incubator with 5% CO₂. Twenty-four hours after transfection, the cells were treated with GC1 or vehicle for an additional 48 h, followed by reporter assays with the EscAPe SEAP Detection Kit according to the manufacturer's instructions (Clontech).

Viral plasmid construction and lentivirus generation.

The lentiviral pSSFV-THRβ1 plasmid was generated by replacing GFP with a PCR product of human THRβ1-6xhis using the Sgf1 and Pmel restriction sites of the pRRLsin-cPPT-SFFV-GFP-wpre vector. Lentiviral particles were generated by cotransfection of pSSFV-THRβ1 with Pax2 and VSVG plasmids in 293T cells (50).

Electrophoretic mobility shift assay.

293T cells were infected with lentiviral particles to express THRβ1 for nuclear protein extraction as previously described (50, 52). Double-stranded DNA probes were labeled with [³²P]γ-ATP at the 5′ ends (46). Nuclear extracts (4 μg) were incubated in a binding buffer containing 10 mM Tris·HCl (pH 7. 9), 50 mM NaCl, 3 mM DTT, 10% glycerol, 0.05% NP-40, 50 μg/ml poly(dI-dC), and 20 fmol of labeled DNA probe. Excess unlabeled DNA competitors were added to the reaction 5 min before addition of the radiolabeled probe. The reactions were incubated at room temperature for 20 min, and then anti-His antibody or control IgG was added to the reactions. The reactions were incubated at room temperature for another 15 min and analyzed on 4% nondenaturing polyacrylamide gels in 0.5× TBE buffer (50 mM Tris-borate-EDTA, pH 8.0). Gels were dried and visualized by autoradiography. Probe sequence: probe: 5′-CCCCCAAAGTCAAAATAGGAGTCAAAATGACCTCAAT-3′. Consensus TRE competitor: 5′-AGCTTCAGGTCACAGGAGGTCAGA. Nonspecific competitor: AACACAAACACAAAGGCG CGG. (Underlined sequences represent consensus TRE.)

Statistical analysis.

Data are presented as means ± SE from 6–10 mice for each group. Significant difference was determined as P ≤ 0.05 or P ≤ 0.01. Data were analyzed by Student's t-test or two-way ANOVA as appropriate. Because we used prepubertal mice (21 days or younger) for all of our experiments and because sex differences become manifested only after puberty (∼5–6 wk of age), we pooled data from both sexes in all of our analyses.

RESULTS

TH stimulates THRβ1 expression in epiphyseal chondrocytes. To identify which of TH receptors is altered in the epiphysial chondrocytes when TH levels are increased, we measured THRα1 and -β1 expression levels using RNA extracted from the epiphysis of the distal femur and the proximal tibia of TH-deficient mice after two daily injections of T₃/T₄ or vehicle. As shown in Fig. 1, A and B, TH treatment caused a more than threefold increase in THRβ1 expression, but had no effect on THRα1 expression compared with vehicle control. Consistent with the real-time PCR data, immunohistochemistry staining with specific antibody against THRβ detected increased THRβ1 protein in the nuclei of SOC chondrocytes (Fig. 1C). To test whether the increase in THRβ1 expression seen in TH treated mice was a direct effect, we treated serum-free primary cultures of epiphysial chondrocytes with 10 ng/ml T₃ for 24 or 72 h. Consistent with the data from the epiphyses of the TH-treated mice in vivo, we found that T₃ treatment caused a significant increase in the THRβ1 mRNA level as early as 24 h in primary chondrocytes (Fig. 1, D and E). At 72 h, the increase was more than 10-fold compared with vehicle-treated control cells. By contrast, there was no increase in THRα1 expression in epiphysial chondrocytes compared with vehicle-treated cells. Treatment with T₃ but not GC1, a THRβ1 specific agonist, increased THRβ1 expression in epiphysial chondrocytes (Fig. 1F), thus suggesting that THRα signaling is involved in the T₃-induced increase in THRβ1 expression in the epiphysis during the postnatal growth period.

Fig. 1.

T₃ treatment upregulates thyroid hormone receptor (THR)β1, but not THRα1 expression in epiphysial chondrocytes. A and B: THRα1 and THRβ1 expression levels in epiphyses of mice. TH-deficient newborn mice at 5 days old were replaced with combination of T₃/T₄ [1 μg triiodothyronine (T₃) and 10 μg thyroxine (T₄)] for 2 days (days 5 and 6), followed by RNA extraction from epiphyses of the distal femur and proximal tibia for real time PCR at day 7 (n = 6, 3 males and 3 females). C: T₃/T₄ treatment stimulates THRβ1 expression in epiphyses of mice described in A and B. Arrows indicate nuclear staining of THRβ1 protein. D and E: THRα1 and THRβ1 expression levels in epiphysial cultures (n = 6, 3 males and 3 females). Epiphyses of distal femur and proximal tibia were cultured and treated with T₃ for 24 and 72 h, respectively, followed by RNA extraction and real-time PCR. Results are expressed as fold change vs. expression level of corresponding vehicle-treated controls. F: THRβ1 expression is stimulated by 48 h with T₃ (10 ng/ml) but not by treatment with THRβ1-specific agonist GC1 (0.1 nM) in primary epiphysial chondrocytes. Values are means ± SE (n = 5, 3 males and 2 females). *Significant difference (P < 0.01) of mRNA expression levels in TH-treated mice or cultures vs. vehicle-treated mice or cultures.

THRβ1 regulates secondary ossification center formation. Since THRβ1 is highly expressed in epiphyseal chondrocytes when the TH level is elevated, we next addressed whether the TH effect on secondary ossification in the epiphysis is mediated via THRβ1. TH-deficient Thsr^−/− mice were treated with GC1, at 0.2 or 2 ug/day at day 5 (Fig. 2A). After two injections, a group of mice were euthanized for RNA extraction and gene expression studies at day 7. Another group of mice were given daily injections for 10 days (day 5–14) and euthanized at day 21 for μCT analyses. As seen from the Fig. 2, B–F, GC1 treatment at both doses caused a more than twofold increase in trabecular bone volume adjusted for tissue volume (BV/TV) that was caused by increases in trabecular number (Tb.N.) and thickness (Tb.Th.) and a reduction in trabecular separation (Tb.S.). After ten days of treatment of Tshr^−/− newborns with the THRβ1 agonist, GC1 at 0.2 or 2.0 μg/day, BV/TV increased by 225 and 263%, respectively, compared with vehicle treatment. Tb.N. was increased by 20–30%, and Tb.Th. was elevated by 29% at a dosage of 0.2 μg/day in the GC1-treated mice. Tb.S. was decreased by 10 and 20% at a dosage of 0.2 and 2 μg/day, respectively. To address the mechanism for the GC1 effect, we measured expression levels of genes that have been implicated in endochondral ossification (47). Two days of GC1 treatment significantly increased expressions of Ihh Osx, Ocn, and Col10 in the epiphyses. Treatment with GC1 (0.2 μg/day) increased expression levels of Ihh by 100-fold, Osx by 15-fold, Ocn by 59-fold, and Col10 by 240-fold compared with vehicle controls at day 7 in the proximal tibia epiphyses. However, expression of the chondrocyte marker gene Col2α1 was decreased by 25% (Fig. 3, A–E).

Fig. 2.

Treatment of TH-deficient mice with THRβ1-specific agonist GC1 promotes epiphysial bone formation. A: schematic diagram of GC1 treatment and tissue harvest. Five-day-old TH-deficient thyroid-stimulating hormone receptor-deficient (Tshr^−/−) mice were treated with 2 μg GC1 for 2 days (days 5 and 6) and euthanized on day 7 for RNA extraction or treated with 0.2 or 2.0 μg GC1 for 10 days from day 5 to day 14, followed by micro-CT analyses at day 21, as indicated in the diagram. B: μ-CT images of epiphyses of proximal tibia treated with GC1 or vehicle for 10 days, from day 5 to day 14. C–F: quantitative μCT data of trabecular bone volume to total volume (Tb.BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp) of tibia epiphyses as shown in B. Values are means ± SE (n = 8, 4 males, and 4 females). *Significant difference (P < 0.01) in Tshr^−/− mice treated with GC1 vs. vehicle-treated Tshr^−/− mice.

Fig. 3.

GC1 treatment of TH-deficient Tshr^−/− mice stimulates Ihh and Osx expression in epiphysial chondrocytes and induces chondrocyte differentiation. A–E: Indian hedgehog Ihh, osterix (Osx), osteocalcin (Ocn), and collagen 10α1 (Col10a1) mRNA levels are induced in the epiphyses of Tshr^−/− mice by 2.0 μg GC1 treatment for 2 days. By contrast, expression of collagen 2 (Col2) is reduced. *Significant difference (P < 0.01) in GC1-treated Tshr^−/− mice vs. vehicle-treated controls in Fig. 2A. Values are means ± SE (n = 5, 3 females, and 2 males). Mean C_T (cycle of threshold) values for the genes in the epiphyses of vehicle-treated Tshr^−/− mice are as follows: Ihh, 28.6; Osx, 26.9; Ocn, 28.1; Col10, 24.8; and Col2, 14.1.

TH induction of Ihh transcription is medicated by THRβ1.

Since we and others have shown that IHH signaling is critically important in endochondral ossification (23, 47), we next focused on the mechanism for the TH effect on Ihh expression. Because of the rapid effect of T₃ on Ihh expression, we predicted a direct effect of TH on Ihh transcription. We therefore searched for the presence of TREs and found at least one TRE located in the distal promoter region of the mouse Ihh gene (Fig. 4A). Similar TREs could also be found in the corresponding regions of rat and human Ihh genes. To evaluate whether THRβ1 interacts with the TRE, we first expressed the THRβ1-his fusion protein in 293T cells by lentiviral infection. Our Western blot analyses with anti-his antibody revealed that THRβ1-his fusion protein was highly expressed in transduced 293T cells and could be detected in 20 μg of nuclear extract (data not shown). We next performed a gel shift assay, and found that the THRβ1-his fusion protein expressed in 293T cells bound to the Ihh TRE (Fig. 4B). This DNA-protein interaction was specific since the complex was competed away with a 200× molar excess of cold Ihh TRE, partially competed with a 200 molar excess of a consensus TRE that THRα1 preferentially binds to, but not by the same molar excess of nonspecific double-stranded DNA oligonucleotides. In addition, the protein-DNA complex was supershifted by 1 μg of antibody specific to the TRβ1-his fusion protein but not by the same concentration of mouse IgG.

Fig. 4.

TH stimulates Ihh transcription via THRβ1 binding to the TRE in the Ihh promoter. A: putative TREs in distal promoters of Ihh are conserved among species in mice, rats, and humans. B: THRβ1-his binds to the TRE of the mouse Ihh gene. EMSA was conducted using 4 μg of nuclear extract from 293T cells overexpressing the THRβ1-his fusion protein with a ³²P-labeled TRE probe. C: GC1 stimulates secreted alkaline phosphatase (SEAP) activity of the pTAL-Ihh SEAP reporter. A portion of the mouse Ihh promoter containing the TRE was inserted in front of the TK minimal promoter of the pTAL-SEAP vector to generate the pTAL-Ihh SEAP reporter. ATDC5 cells were transfected with the reporter constructs and then treated with 100 nM, 1 nM GC1, or vehicle. Alkaline phosphatase activity was analyzed 48 h after treatment and expressed as fold changes compared with vehicle-treated cells. D: Ihh mRNA expression is stimulated in epiphysial cultures of Tshr^−/− mice by 3 days of GC1 treatment, measured by real-time PCR. Values are means ± SE (n = 6, 3 females, and 3 males). *Significant difference in GC1-treated cells (P < 0.01) vs. corresponding vehicle-treated cells.

To determine whether the TREs found in Ihh promoter were functional, we inserted a 1-kb portion of the mouse Ihh promoter in front of the TK minimal promoter of the pTAL-SEAP reporter and performed a reporter assay. We found that GC1 treatment at 100 nM and 1 nM final concentrations increased reporter activity 12- and 20-fold, respectively, compared with vehicle treatment at 48 h in ATDC5 chondrocytes (Fig. 4C). By contrast, the same GC1 treatment of the cells transfected with the promoterless construct pTAL-SEAP did not induce SEAP activity. Consistent with the reporter assay, 48 h of GC1 treatment of primary epiphysial chondrocytes increased Ihh expression 1.8-fold, as evidenced by real-time PCR analyses (Fig. 4D).

DISCUSSION

Previous immunohistochemistry studies have shown that THRα1 and -β1 were localized in the nuclei of reserve zone progenitor cells, proliferating chondrocytes, and osteoblasts but were absent from the hypertrophic chondrocytes of tibia growth plates of mature rats (41). While THRα1 expression was widespread in all cells throughout these regions, THRβ1 staining was weak and present only in a subset of cells. Consistent with these data, our data showed that THRα1 was constitutively expressed at higher levels in the epiphysis throughout the postnatal growth period. By contrast, THRβ1 expression was reduced in hypothyroid mice and that treatment with TH caused a severalfold increase in THRβ1 expression in epiphysial chondrocytes both in vitro and in vivo. In terms of mechanisms for TH-induced increases in THRβ1 expression, it is known that the THRβ1 gene promoter contains TREs and is a TH-responsive gene (9). Accordingly, a recent study demonstrated a repressive role of unliganded THRα1 in regulating expression of several TH-responsive genes, including THRβ1, during amphibian metamorphosis (36, 38). While these data are suggestive that a prepubertal increase in TH during the second week of postnatal development in mice results in the loss of repressive action of the unliganded THRα1 on THRβ1 promoter to promote transcription, further experiments are needed to confirm this mechanism for the TH-mediated increase in THRβ1 expression in epiphysial chondrocytes during the prepubertal growth period.

Studies in knockout and mutant mice with disrupted TH signaling have revealed that THRα1 is the functionally predominant THR isoform expressed in cartilage and bone (13, 44), and the recent identification of patients with autosomal dominant mutations affecting THRα1 are consistent with an important role for THRα1 in skeletal development and postnatal growth (26, 43). THRβ^−/− mice lacking all forms of THRβ display features of skeletal hyperthyroidism with consistent short stature and advanced endochondral and intramembranous ossification during development (5, 12). By contrast, adult THRβ^−/− mice have osteoporosis with reduced trabecular bone volume (30, 32). However, since these mice have elevated T₄, T₃, and TSH levels, it is difficult to ascertain whether the observed skeletal phenotype is due to superphysiological activation of normal and predominant THRα1 in bone by elevated circulating T₃ and T₄ levels and/or is due to loss of THRβ. Our findings that expression of THRβ1 was increased severalfold in response to TH treatment in the epiphysis suggested a role for this receptor in secondary ossifications of the epiphysis. To evaluate the role of THRβ1 in the secondary ossification process, we treated mice with GC1, a THRβ1-specific agonist (18) during the time when serum T₃ levels were elevated in mice. Our findings demonstrated that treatment of TH-deficient mice with GC1 for 10 days caused a dramatic increase in new bone formation in the epiphyses of mice, thus suggesting a positive role of THRβ1 in promoting secondary ossification at the epiphysis. Consistent with this finding, a previous study showed that GC1 treatment of hypothyroid mice for 5 wk increased trabecular bone volume in the femur and tibia (14). Future studies involving chondrocyte-specific disruption of THRβ1 during the second week of the postnatal growth period, when THRβ1 expression is increased, are necessary to confirm a physiological role for THRβ1 in the ossification of the epiphysis.

In terms of the mechanism by which GC1-mediated activation of THRβ1 promotes endochondral ossification at the epiphysis, we found that expression levels of several growth factors and transcription factors previously implicated in endochondral ossification were highly induced in the epiphysis of GC1-treated hypothyroid mice (47). We focused on THRβ1-mediated regulation of the Ihh gene, since our previous studies as well as other studies had indicated a key role for IHH signaling in promoting endochondral ossification. Ihh-null embryos exhibit severe defects in chondrocyte development and fail to form osteoblasts, resulting in neonatal lethality (39), while inducible deletion of Ihh in chondrocytes in newborn mice caused growth plate disruption and trabecular bone loss at later stages (23). Targeted overexpression of Ihh in Col2-expressing chondrocytes resulted in postnatal lethality and blockade of chondrocyte differentiation at the prehypertrophic stage. Furthermore, chemical inhibition of hedgehog signaling with cyclopamine in 8-wk-old male mice reduced the bone mass due to decreased osteoblast number and function (31). These and other data suggest that the effect of IHH signaling is dependent on the level or duration of the signal, the target cell type, as well as the systemic factors.

Gene regulation by TH is achieved through activation of the THR, which is bound to TREs in the regulatory regions in the presence or absence of ligand. THRs repress expression of TH-responsive genes in the absence of T₃ and activate their target genes when T₃ is available by recruiting corepressors and coactivators, respectively (32, 38). These dual functions suggest that both unliganded and T₃-bound THRs affect normal and pathological processes in vivo. TREs are generally composed of two half-sites, with AGGTCA hexamer being the classic half-site (32). The orientation and spacing of the half-sites vary across TREs. Of the three well-known functional TREs [direct repeat 4 (DR4), inverted repeat 0 (IR0), and everted repeat 6 (ER6)], the Ihh promoter from different species contains an ER6-type TRE (33, 34). It has been shown that homodimers of THRβ preferentially bind to and regulate genes possessing ER6 TREs (4, 8, 28). Since the Ihh gene contains an ER6 TRE, and since THRβ has a preferential binding to the ER6 type TRE, we examined whether the TH effect on Ihh transcription was mediated via THRβ1 binding to the ER6-type TRE in the distal promoter region. Our data show that THRβ1 interacts with the TRE with six nucleotides spacing in the Ihh promoter and promotes transcription in the presence of TH ligand. Whether other transcription factors interact with THRβ1 and are involved in mediating TH effects on Ihh transcription will need to be identified in order to establish the precise molecular mechanism of TH regulation of Ihh expression.

In conclusion, we predict, on the basis of our data and other published data, that the increase in circulating levels of T₃ and T₄ during the prepubertal growth period bind to THRα1 to relieve its suppressive effect on THRβ1 expression in epiphysial chondrocytes. THRβ1 in the presence of T₃ stimulates Ihh transcription and leads to transdifferentiation of epiphysial chondrocytes into osteoblasts and thereby stimulates endochondral bone formation in the epiphysis.

GRANTS

This research was supported by National Institutes of Health Grant AR-048139 to S. Mohan and a Senior Research Career Scientist award to S. Mohan from the Veteran's Administration. The funder had no role in study design, data collection, or analysis, decision to publish, or manuscript preparation. The research work was performed at facilities provided by the Department of Veterans Affairs.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: W.X. and S.M. conception and design of research; W.X., P.A., H.G., C.K., S.C., S.P., H.W., and C.A. performed experiments; W.X. and S.M. analyzed data; W.X. and S.M. interpreted results of experiments; W.X. and S.M. prepared figures; W.X. and S.M. drafted manuscript; W.X. and S.M. edited and revised manuscript; W.X. and S.M. approved final version of manuscript.